ZF21 is a new regulator of focal adhesion disassembly and a potential member of the spreading initiation center

Abstract

Adherent cells migrate on extracellular matrices (ECM) by repeated spreading and contraction of the cell body. Focal adhesions (FAs) play a major role in the adherence of cells to the ECM and in the generation of the cellular forces that maintain morphology and allow cells to move. FAs also mediate bidirectional transmembrane signals in conjunction with growth factor receptors and signaling molecules. Although the mechanisms that regulate cell migration are not yet fully understood, the regulation of the formation and turnover of FAs is a key factor determining the rate and direction of cell migration. We recently identified a component of FAs termed ZF21, which is a member of a family of proteins characterized by the presence of a conserved phosphoinositide-binding motif. ZF21 promotes dephosphorylation of FAK at Tyr ( 397) upon microtubule extension to FAs and thereby regulates the disassembly of FAs in a microtubules-dependent manner. To obtain further insight into the regulation of cell adhesion by ZF21, we analyzed proteins associating with ZF21 by proteomic analysis. We identified 45 proteins including FA-related proteins and multiple RNA binding proteins that have been shown recently to be components of the spreading initiation center (SIC). SICs are cell adherent structures that can be observed only in the early stages of cell spreading and have been implicated in regulating the rate of cell spreading. In this article, we report new ZF21-binding proteins identified by proteomic analysis and discuss the potential functions of ZF21 in regulating disassembly of FAs.

Figure 1

Analysis of proteins associating with ZF21. (A) ZF21 fused to GST was used to pull down proteins from whole cell lysates of HeLa cells as described previously. Proteins specifically bound to GST-ZF21 were analyzed by western blot using antibodies against the indicated proteins (FAK, talin, vinculin or GST). (B) The proteins pulled down using GST-ZF21 or GST-EGFP were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The protein bands that appeared to bind specifically to GST-ZF21 but not to GST-EGFP were cut out and these bands were subjected to in-gel digestion with trypsin and analyzed by LC/MS. Controls were the corresponding gel fragments in GST-EGFP column. Asterisks indicate GST-EGFP and GST-ZF21 proteins.

Figure 2

A possible mechanism of ZF21-mediated disassembly of FAs. (A) Domain structure of GST-ZF21 derivatives used for the pull-down assays in (B). Full (1–234 aa), full-length; N (1–105 aa), an N-terminal domain fragment including FYVE domain; C (106–234 aa), a C-terminal domain mutant. The amino acid positions are indicated by aa. (B) Pull-down experiments were carried out as described in Figure 1A. The proteins bound to GST-ZF21 derivatives were subjected to western blot analysis using antibodies against FAK, m-calpain, b-tubulin or GST. (C) Binding regions of ZF21 with FAK, m-calpain or b-tubulin are summarized based on the results in (B). (D) A model proposed for ZF21-mediated disassembly of FAs, though association of proteins with ZF21 may not be direct. ZF21 resides in FAs even when MT extension to the FA is disrupted by nocodazole treatment. ZF21 binds to FAK in FAs via its FYVE domain. The C-terminal region of the pre-existing ZF21 in FAs may bind MTs that have extended to the FAs. ZF21 may also reside in the transport vesicles via the FYVE domain. Since ZF21 can bind SHP-2 and m-calpain, these factors might also be transported to FAs by MTs if ZF21 interacts with the vesicles transported by kinesin-1 on MTs. Binding of m-calpain to ZF21 requires the entire region of the protein, but the binding site of ZF21 to SHP-2 is not known. The pre-existing ZF21 in FAs may form oligomers with the ZF21 transported to the FAs by vesicles. The ability of ZF21 to form oligomers in FAs may bring phosphorylated FAK in closer proximity to SHP-2. m-Calpain cleaves integrin, FAK, talin, paxillin and vinculin. The ZF21 oligomer also acts as a platform to accumulate factors for executing the disassembly of FAs. Thus, MT extension to FAs causes accumulation of factors responsible for the initiation of the disassembly of FAs. Finally, the remains of the disassembled FAs are internalized in a dynamin-dependent manner.